Anti cd31 antibody

After isolation of BM cells from donor mice, flow cytometric analysis was performed to compare the BM cell populations of the BMT and c‐BMT methods. After lysing RBCs with ammonium chloride, cells were filtered through a cell strainer (100 μm, BD Falcon). Cells (1.0 × 10⁷) were suspended in 100 μL of 2% FBS/PBS and then incubated with anti‐leptin receptor (LepR) biotinylated antibody (R&D Systems, Minneapolis, MN, USA; BAF497, 1:100), anti‐CD51 antibody (BioLegend, San Diego, CA, USA; 104105, 1:100), anti‐CD45 antibody (BioLegend, 103111, 1:100), anti‐TER‐119 antibody (BioLegend, 116211, 1:100), and anti‐CD31 antibody (BioLegend, 102509, 1:100) for 60 minutes on ice. Then cells were washed and incubated with streptavidin‐BV421 antibody (BioLegend, 405226, 1:1000) for 20 minutes on ice. For RUNX2 detection, cells were fixed and permeabilized with a transcription factor buffer set (BD Biosciences, San Jose, CA, USA) and incubated with an anti‐RUNX2 antibody (Cell Signaling, Danvers, MA, USA; #12556 S, 1:200) for 30 minutes on ice. Then cells were washed and incubated with anti‐rabbit antibody (Cell Signaling, #4412 S, 1:400) for 30 minutes on ice. Fluorescence was detected using a MACSQuant (Miltenyi Biotec, Bergisch Gladbach, Germany). Data analysis was performed using FlowJo software (BD Biosciences).